Lab Report: Isolation of Pure Culture, Gram-staining, and Microscopic Observation

A Lab Report under the subject of Microbiology. Done as a lab session in Josai University, Japan during a
twinning program on 2014.

Created by: Annisa Hayatunnufus

 

Bachelor of Pharmacy
Management & Science University

 Lab Report: Isolation of Pure Culture, Gram-staining, and Microscopic Observation

1. 1. PHARMACEUTICAL SCIENCES Subject: MICROBIOLOGY Lab Report: Isolation of Pure Culture,

Gram-staining, and Microscopic Observation Prepared by: ANNISA HAYATUNNUFUS ID number:
012014052438 (MSU) – PF14012 (JOSAI) Lecturer’s Name : ProfessorKondoSeiichi Date of
submission :
 

2. 2. 2 CONTENT I. Objectives..............................................................................3 II.

Introduction.................................................................................................................3 III.
Materials & Apparatus.......................................................................................5 IV.
Procedures ..........................................................................5 V.
Results .................................................................................7 VI.
Discussion ...........................................................................8 VII.
Reference.........................................................................10
 

3. 3. 3 I. OBJECTIVES 1. Isolating pure culture from contaminated sample and extracting an
independent single colony. 2. Classifying microorganisms into gram-positive or gram-negative
through staining reaction in order to distinguish the permeability of its cell wall for selection of
anti- bacterial agent. 3. Determination of microorganism’s characteristics by microscopic
observation. 4. Identification of microorganism species from obtained characteristics. II.
INTRODUCTION Staining is an auxiliary technique used in microscopic techniques that have the
function to enhance the clarity of the microscopic image. Stains and dyes are widely used in the
scientific field to highlight the structure of the biological specimens, cells, tissues etc. The most
 

widely used staining procedure in microbiology is the Gram stain, discovered by the Danish
scientist and physician Hans Christian Joachim Gram in 1884. Gram staining is a differential
staining technique that differentiates bacteria into two groups: gram-positives and gram-
negatives. The procedure is based on the ability of microorganisms to retain color of the stains
used during the gram stain reaction. Gram-negative bacteria are decolorized by the alcohol,
losing the color of the primary stain, purple. Gram-positive bacteria are not decolorized by
alcohol and will remain as purple. After decolorization step, a counterstain is used to impart a
 

pink color to the decolorized gram-negative organisms. Figure 2.1. Colour changes that occur at
each step in the staining process

4. 4. 4 Despite the major classification of these two groups, several bacteria are classified as the
gram-indeterminate bacteria. Also known as gram-variable bacteria, this type of bacteria do not
respond predictably to Gram staining and, therefore, cannot be determined as either gram-
positive or gram-negative. They tend to stain unevenly, appearing partially gram positive and
partially gram negative, or even unstained. Staining older cultures (over 48 hours) can lead to
false gram-variable results, probably due to changes in the cell wall with aging. Gram-
 

indeterminate bacteria are best stained using acid-fast staining techniques. Examples include
many species of Mycobacterium, including M. tuberculosis and M. leprae. The Gram stain is a
very important preliminary step in the initial characterization and classification of bacteria. It is
also a key procedure in the identification of bacteria based on staining characteristics, enabling
the bacteria to be examined using a light microscope. The bacteria present in an unstained
smear are invisible when viewed using a light microscope. Once stained, the morphology and
arrangement of the bacteria may be observed as well. Furthermore, it is also an important step
in the screening of infectious agents in clinical specimens such as direct smears from a patient.
 

The samples of microorganisms that are going to be tested are Escherichia coli, Stapylococcus
aureus, and Candida albicals. E.coli (rod-shape) and S.aureus (spherical form) are both bacteria.
Meanwhile, Candida albicals (yeast or mycelial form) is a fungi and with S.aureus, both are
commonly known as a gram-positive microorganism. Hence, in the sample, E.coli will be the only
microorganism that is classified as gram-negative.

5. 5. 5 III. MATERIALS & APPARATUS MATERIALS APPARATUS Artificially mixed suspension of
bacteria & fungus (Escherichia coli, Stapylococcus aureus, Candida albicals) Simple general tools
(tissue, hand gloves, etc.) Solid medium (Agar plate) Simple laboratory tools (test tubes, racks,
 

pipettes, gas burner, etc.) Sterilized saline Incubator Crystal violet solution Platinum loop Lugol
solution Platinum needle Ethanol Filter paper Fuchsine solution Slide glass Distilled water
Microscope, lens cleaner, immersion oil IV. PROCEDURES 1. Isolation of Bacterial Strain (Pure
Culture) from Mixed Specimen - Sterilize the platinum loop and cool it down. - Take one loop of
bacterial suspension. - Spread on the solid medium (agar plate) as shown on figure 4.1.1. -
Sterilize the platinum loop and cool it down. Spread once more on the solid medium (agar plate)
as shown on figure 4.1.2. - Sterilize the platinum loop again, and then cool it down. Spread one
last time on the solid medium (agar plate) as shown on figure 4.1.3. - Sterilize the platinum loop,
 

cool it down, and store it back. Meanwhile, close the medium with its lid and incubate for
appropriate hours at 37ºC. - Once the process is done, the medium will be similar to figure 4.1.4.
Figure4.1.1.Figure4.1.2.Figure4.1.3.Figure4.1.4.

6. 6. 6 2. Gram-staining: Preparation of Smear - Take one loop of sterilized saline on a slide glass. -
Take a small amount of bacterial cells from independent single colony using platinum needle. -
Suspend the bacterial cells in saline and extend the smear circle approximately 1.5 cm diameter.
- Dry at room temperature. - Fixation of bacterial cells onto the surface of slide glass by heating
(passing the slide glass through the flame of the gas burner). - Cool down the slide glass to the
 

room temperature. 3. Gram-staining: Staining - Apply 4 to 5 drops of crystal violet solution

(Gram-staining solution no.1) on the smear for 2 minutes. - Wash off the crystal violet solution
by adding Lugol solution (Gram-staining solution No.2) - Apply 4 to 5 drops of the Lugol solution
for 2 minutes. - Discard the Lugol solution and decolourize with absolute ethanol for 1 to 1.5
minutes. - Wash the back side of the slide glass with distilled water. - Apply 4 to 5 drops of
fuchsine solution (Gram-staining solution No.3) for 1 minute. - Wash the back side of the slide
glass again with distilled water. - Remove excess water using filter paper and dry the slide glass.
4. Microscopic observation
 

7. 7. 7 V. RESULTS SMEAR 1 SMEAR 2 SMEAR 3

8. 8. 8 VI. DISCUSSION I. THEORY The Gram stain procedure enables bacteria to retain color of the
stains, based on the differences in the chemical and physical properties of the cell wall. 1. Gram
positive bacteria Stain dark purple due to retaining the primary dye called Crystal Violet in the
cell wall. Gram-positive bacteria have a thick mesh-like cell wall which is made up of
peptidoglycan (50-90% of cell wall), which stains purple. The thick peptidoglycan layer of Gram-
positive organisms allows these organisms to retain the crystal violet-iodine complex and stains
the cells as purple (see figure 6.1.) 2. Gram negative bacteria Stain red or pink due to retaining
 

the counter staining dye called Safranin. Gram-negative bacteria have a thinner layer of
peptidoglycan (10% of the cell wall) and lose the crystal violet-iodine complex during
decolorization with the alcohol rinse, but retain the counter stain Safranin, thus appearing
reddish or pink. They also have an additional outer membrane which contains lipids, which is
separated from the cell wall by means of periplasmic space (see figure 6.2.) Figure 6.1. Figure
6.2.

9. 9. 9 II. IDENTIFICATION 1. SMEAR 1: a. Smear number 1 has a pinkish color, which classify itself
as a gram-negative microorganism. b. It has the shape of a rod with identical characteristics for
 

the whole colony and that it resembles the shape and characteristic of the bacteria in smear 2.
c. It has the same size as smear 2, but slightly bigger than smear 3. d. Smear 1, same as smear 2,
is Escherichia coli. 2. SMEAR 2: a. Smear number 2 also has a pinkish color, which made it a
gram-negative microorganism. b. It has the shape of a rod with identical characteristics for the
whole colony and that it resembles the shape and characteristic of the bacteria in smear 1. c. It
has the same size as smear 1, but slightly bigger than smear 3. d. Smear 1, same as smear 2, is
Escherichia coli. 3. SMEAR 3: a. Smear number 3 has a deep purple color, which classify itself as
a gram- positive microorganism. b. It has a circular/spherical shape with identical characteristics
 

for the whole colony. c. It is slightly smaller compared to smear 1 and 2. d. Smear 3 is
Stapylococcus aureus.

10. 10. 10 VII. REFERENCES  WEBLIOGRAPHY 1. http://amrita.vlab.co.in/?

sub=3&brch=73&sim=208&cnt=6 2. http://en.wikipedia.org/wiki/Gram_staining  TEXT BOOK 1.
Black, Jacquelyn (2012). Microbiology: Principles and exploration 8th edition. John Wiley & Sons.
p. 68. ISBN 978-0-470-54109-8 (via Wikipedia)

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